Journal: Pharmacological Reports

Article Title: Targeting mitochondrial deubiquitinase USP30 to induce mitophagy in heteroplasmic mitochondrial diseases

doi: 10.1007/s43440-026-00829-7

Figure Lengend Snippet: Mutant cells exhibit reduced proliferation and ATP levels under OXPHOS-dependent conditions and resistance to mitochondrial inhibition under glycolytic conditions. 143B cybrid cells harboring 0% (N0), 40% (N40) or 80% (N80) of the m.8993T > G mtDNA mutation were cultured under glycolytic (glucose-containing medium) or OXPHOS-dependent conditions (galactose-containing medium – without glucose). (A) Confirmation of the mutant mtDNA load by ARMS-PCR. Data are mean ± SEM, n = 4 independent experiments (B) Mitophagy of N0 and N80 cells under glycolytic conditions was assessed by live imaging of the mitochondrial protein COX8 linked to both EGFP and mCherry: (i) schematic representation of the dual-fluorescent mitochondrial protein COX8-EGFP-mCherry: at neutral/alkaline pH, both EGFP and mCherry fluoresce, while at acidic pH (e.g. in mitolysosomes), only mCherry fluorescence is retained; (ii) representative images – red dots identify mitolysosomes (mCherry fluorescence only); (iii) quantification of the number of mitolysosomes per cell. n = 312–318 cells, from 3 independent experiments. Results are presented as median and interquartile range, with extremes presenting 10–90 percentiles, * p < 0.05, Mann-Whitney test. C) Cellular proliferation: growth over time after seeding in (i) glucose- or (ii) galactose-containing medium; (iii) representative images at 72 h after seeding. Initial cell density was similar across all conditions. Data are mean ± SEM, n = 5–7 independent experiments, non-linear regression following exponential growth equation. We used extra sum-of-squares F test for statistical comparison of growth curves: a single curve fits all data under glycolytic conditions (glucose), but not in OXPHOS-dependent conditions (galactose). D) ATP levels and ATP/ADP ratio quantified by HPLC of cells cultured in (i) glucose- and (ii) galactose-containing medium. Data are mean ± SEM, n = 3–4 independent experiments; * p < 0.05, one-way ANOVA followed by Dunnett’s multiple comparisons test. E) Resazurin metabolism after treatment of cells cultured in glucose- and galactose-containing medium with increasing concentrations of classical mitochondrial inhibitors for 24 h: (i) rotenone (complex I inhibitor); (ii) myxothiazol (complex III inhibitor); (iii) antimycin (complex III inhibitor); (iv) oligomycin (ATP synthase inhibitor). Data are mean ± SEM in % of N0 without drug treatment; n = 4 independent experiments

Article Snippet: Cells were seeded in glass-bottom Ibidi chamber slides (Ibidi, 80827) at a density of 15,000 cells/cm 2 , washed with DMEM (Gibco, 31966021), and incubated with a mixture of Lipofectamine LTX (Invitrogen, 15338030), Reagent plus and plasmid DNA (0.5 μg pCLBW-cox8-EGFP-mCherry, Addgene, 78520 [ ]) at a ratio of 2:1:1.

Techniques: Mutagenesis, Inhibition, Cell Culture, Imaging, Fluorescence, MANN-WHITNEY, Comparison

Journal: Pharmacological Reports

Article Title: Targeting mitochondrial deubiquitinase USP30 to induce mitophagy in heteroplasmic mitochondrial diseases

doi: 10.1007/s43440-026-00829-7

Figure Lengend Snippet: MF-094 promotes mitochondrial protein ubiquitination and formation of mitolysosomes. A) Quantification of MF-094 by UV-spectrophotometry: (i) UV spectrum of increasing concentrations of MF-094, showing an absorbance peak at 308 nm; (ii) calibration curve of MF-094: absorbance at 308 nm as a function of concentration; (iii) UV spectrum of 30 µM MF-094 after 24, 48, and 72 h of incubation under cell culture conditions in presence of cells; (iv) quantification of MF-094 in cell culture medium, with and without cells (initial concentration: 30 µM). Results from 3 independent experiments are presented as mean ± SEM. B) Metabolism of resazurin in cells treated with increasing concentrations of MF-094 under (i) glycolytic (glucose) or (ii) OXPHOS-dependent conditions (galactose); results from 3 to 4 independent experiments are expressed as mean ± SEM, as percentage of N0 cells without drug treatment. C) Mitochondrial protein ubiquitination was assessed by western-blot using an antibody against ubiquitin and a mitochondria-enriched extract from N80 cells treated with increasing concentrations of MF-094 under glycolytic conditions for 2 h: (i) representative blot and (ii) respective quantification. n = 6 independent experiments. D) TOM20 ubiquitination after N80 cell treatment with increasing concentrations of MF-094 under glycolytic conditions for 2 h: (i) representative blot – the panel identified with “high exposure” highlights ubiquitinated TOM20 (higher molecular weight than non-ubiquitinated TOM20); (ii) ubiquitinated TOM20 quantification. n = 6 independent experiments. C , D) Results are presented as mean ± SEM, * p < 0.05, one-way ANOVA with Dunnett’s multiple comparisons test. E) Mitophagy of N80 cells treated with 10 µM MF-094 under glycolytic conditions for 24 h was assessed by live imaging of the mitochondrial protein COX8 linked to both EGFP and mCherry: (i) representative images – red dots identify mitolysosomes (mCherry fluorescence only); quantification of the (ii) number and (iii) average area of mitolysosomes per cell. The inset graphs show amplified images of the regions of interest in the main graphs. n = 349–385 cells, from 4 independent experiments. Results are presented as median and interquartile range, with extremes presenting 10–90 percentiles, * p < 0.05, Mann-Whitney test

Article Snippet: Cells were seeded in glass-bottom Ibidi chamber slides (Ibidi, 80827) at a density of 15,000 cells/cm 2 , washed with DMEM (Gibco, 31966021), and incubated with a mixture of Lipofectamine LTX (Invitrogen, 15338030), Reagent plus and plasmid DNA (0.5 μg pCLBW-cox8-EGFP-mCherry, Addgene, 78520 [ ]) at a ratio of 2:1:1.

Techniques: Ubiquitin Proteomics, Spectrophotometry, Concentration Assay, Incubation, Cell Culture, Western Blot, Molecular Weight, Imaging, Fluorescence, Amplification, MANN-WHITNEY

Journal: bioRxiv

Article Title: The antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction

doi: 10.1101/2025.05.16.654477

Figure Lengend Snippet: (A) NTC or APOL3 -/- HeLa cells primed overnight with IFN-γ, were pulsed with LLOME (600 μM, 2 hr unless otherwise indicated), recovered for 8 h and analyzed by RNAseq. Volcano plots show differential expression between untreated (DMSO) and LLOME treated for both genotypes. Significant genes (P < 0.01, log 2 -fold change (LFC) > 2) are depicted in rose (upregulated) or purple (downregulated). Selected genes of interest were annotated (ISGs in teal). Statistical significance was calculated using the Wald test, with P values adjusted by the Benjamini-Hochberg procedure. (B) NTC or APOL3 -/- HeLa cells expressing pMSCV-empty or pMSCV- APOL3 were pulsed with LLOME and IFN-β protein levels in the supernatants determined by ELISA after 12 hr. (C) HeLa genotypes expressing IRF3-luciferase +/- IFN-γ priming pulsed with LLOME or treated with Poly(I:C) and luminescence measured after 6 hr. Data is fold change relative to mock treated. (D) IFN-β in the supernatants of primed or unprimed NTC or APOL3 -/- HeLa cells after the indicated insult. (E and F) IFN-β (E) or IL-1β (F) in the supernatants of IFN-γ/IL-1β-primed THP-1 macrophages 24 hr after being fed SiO 2 nanoparticles (50 μg/ml for 4 hr) to trigger phagosomal rupture. APOL3 was expressed in trans via lentivirus transduction. (G) IFN-β levels in the supernatants of NTC or APOL3 -/- cells infected (8 hr) with invasive (SPI-1), hyper-invasive (Δ sifA ), or non-invasive (SPI-2) Salmonella Typhimurium ( Stm ), invasive adenovirus (AdV), or non-invasive AdV- ts1 . (H) HeLa genotypes +/- IFN-γ priming infected with AdV-GFP (24 hr) and imaged (representative images shown). Poly(I:C) or anti-IFNAR antibodies were co-incubated for the duration. Percentage values denote mean ± SD, n = 3) (I) IFN-β in the supernatants of IFN-γ-primed HeLa cells of the indicated genotypes 24 hr after LLOME pulse. Representative immunoblots for each genotype are shown. Data represent mean ± SEM from 3 or 4 independent experiments. *P < 0.05; ** P < 0.01; *** P < 0.001 determined by one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: NTC or APOL3 -/- HeLa cells were transfected with pCLBW cox8-EGFP-mCherry (Addgene #78520) a gift from David Chan .

Techniques: Quantitative Proteomics, Expressing, Enzyme-linked Immunosorbent Assay, Luciferase, Transduction, Infection, Incubation, Western Blot, Comparison

Journal: bioRxiv

Article Title: The antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction

doi: 10.1101/2025.05.16.654477

Figure Lengend Snippet: (A) IFN-γ-primed HeLa genotypes expressing FLAG-cGAS or FLAG-GFP were treated with LLOME followed by extraction of DNA from FLAG immunoprecipitants and mitochondrial ( ND1 , ND2 ), nuclear ( L1 , PG ) or ribosomal ( 18S ) transcripts. Fold change (FC) is relative to mock treated. (B) IFN-β produced by IFN-γ-primed APOL3 -/- cells rescued with retroviral- APOL3 and cultured with ddC to deplete mtDNA prior to treatment. (C) qPCR quantification of mtDNA in the cytosol relative to whole cell extract (WCE) after treatment with LLOME or ABT-737/Q. Shown are floating bars (mean, min/max, n = 5 independent experiments) (D) APOL3 mnGFP knock-in cells expressing OMP25 JF646 and Lamp1-mCherry were pulsed with LLOME and imaged at the indicated time. Fractional overlap for APOL3 is shown (n = 25 cells, error bars ± SD). Results are representative of 3 independent experiments. (E) IFN-γ/IL-1β-primed THP-1 macrophages expressing APOL3-GFP were fed SiO 2 particles (50 μg/ml, 3 hr) and immunostained for Tom20 or Lamp1. (F) APOL3 mnGFP cells infected with SPI-1 Stm (DAPI) for 3 hr and mitochondria labeled with OMP25 JF646 . Images are representative single z planes of Thunder deconvolved widefield images. Scale bar, 5 μm. Data are mean ± SEM from 3 (A) or 4 (B) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 determined by two-tailed unpaired Student’s t tests (A, D) or one-way ANOVA with Tukey’s multiple comparison test (B, C).

Article Snippet: NTC or APOL3 -/- HeLa cells were transfected with pCLBW cox8-EGFP-mCherry (Addgene #78520) a gift from David Chan .

Techniques: Expressing, Extraction, Produced, Retroviral, Cell Culture, Knock-In, Infection, Labeling, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: The antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction

doi: 10.1101/2025.05.16.654477

Figure Lengend Snippet: (A, B) IFN-γ-primed HeLa cells expressing cytosolic GFP (CytoGFP) and mitochondrial mCherry (MitoCherry) were treated with LLOME or ABT-737/Q with heterodimerizer and imaged. Total mitochondrial volume (MitoCherry) overlapping with cytoGFP is shown (mean ± SD from 15 (A) or 50 (B) cells. (C) IFN-γ-primed NTC or BAK/BAX -/- cells expressing APOL3-GFP, OMP25 646 , or Lamp1-mCherry treated with LLOME. Fractional overlap for APOL3 is shown (n = 25 cells, error bars ± SD). (D) qPCR of cytosolic mtDNA relative to WCE in IFN-γ-primed NTC or BAK/BAX -/- cells treated with LLOME. (E) IFN-γ-primed BAK/BAX -/- cells as in (C) were treated with LLOME with or without CCCP. Fractional overlap for APOL3 is shown (n = 11 cells, error bars ± SD). (F) qPCR quantification of mtDNA in the cytosol relative to WCE of IFN-γ-primed BAK/BAX -/- cells with the indicated agonist. (G) Representative immunoblots of cytosolic (cyto) or mitochondrial (mito) fractions prepared from IFN-γ-primed HeLa genotypes treated with LLOME or ABT-737/Q. (A), (B), (C), (E) are representative or >3 independent experiments. (D) and (F) are floating bars (mean, min/max, n = 3 or 4 independent experiments). ** P < 0.01; *** P < 0.001 determined by one-way ANOVA. Images are maximum intensity projections (A, B, E) or single z planes (C) of Thunder deconvolved widefield images. Scale bar, 5 μm.

Article Snippet: NTC or APOL3 -/- HeLa cells were transfected with pCLBW cox8-EGFP-mCherry (Addgene #78520) a gift from David Chan .

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: The antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction

doi: 10.1101/2025.05.16.654477

Figure Lengend Snippet: (A) IFN-γ-primed HeLa cells co-expressing the indicated variant of APOL3-GFP and OMP25 JF646 treated with LLOME. Volume of OMP25 overlapping with APOL3 is shown (n = 20 cells, mean ± SD). Images are single z planets of Thunder deconvolved widefield images and are representative of 3 independent experiments. Scale bar, 5 nm. (B and C) IFN-γ-primed APOL3 -/- cells rescued with the indicated APOL3 variant were treated with LLOME (2 hr) and mtDNA in the cytosol relative to WCE determined by qPCR (B) or luminescence detected in cells expressing IRF3-luciferase after an additional 4 hr (C). (D) APOL3 -/- or NTC cells +/- IFN-γ priming rescued with the indicated APOL3 variant and infected with Stm Δ sifA for 6 hr or AdV for 12 hr. FC relative to mock treated for each gene was determined by qPCR (n = 2). (E and F) IFN-γ/IL-1β-primed APOL3 -/- THP-1 macrophages rescued with the indicated APOL3 variant were fed SiO 2 particles to induce phagosomal rupture. Cytosolic mtDNA relative to WCE was determined by qPCR (E), or IFN-β measured by ELISA after 24 hr (F). (B) and (E) are floating bars (mean, min/max, n = 5 independent experiments). (C) and (F) are mean ± SEM from 3 or 4 independent experiments. *P < 0.05; ** P < 0.01; *** P < 0.001 determined by one-way ANOVA.

Article Snippet: NTC or APOL3 -/- HeLa cells were transfected with pCLBW cox8-EGFP-mCherry (Addgene #78520) a gift from David Chan .

Techniques: Expressing, Variant Assay, Luciferase, Infection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction

doi: 10.1101/2025.05.16.654477

Figure Lengend Snippet: (A, B) IFN-γ-primed HeLa cells pulsed with ABT-737 or LLOME with or without pan-caspase inhibition (Q-VD-Oph) and analyzed by Flow cytometry for Annexin-V staining or IFN-β production by ELISA, both after 24 hr. (C) Time course immunoblots of cytosolic or mitochondrial fractions from IFN-γ-primed HeLa cells incubated with LLOME or ABT-737. Blots are representative of 3 independent experiments. (D) IFN-γ-primed NTC or APOL3 -/- cells expressing cytochrome c – GFP and TFAM-mScarlet treated with ABT-737/Q or LLOME. Images are maximum intensity projections, representative of 3 independent experiments. Quantification is mean ± SEM from 3 independent experiments (n = 20 cells per replicate). Scale bar, 5 μm. (E) IFN-γ-primed NTC or APOL3 -/- HeLa cells expressing FLAG-cGAS were pulsed with LLOME (2 hr) at the indicated concentration. 2 hr later cell death was measured by PI uptake (right y axis, rose) followed by qPCR for mtDNA in FLAG immunoprecipitants (left y axis, black). Where indicated treatments included Q-VD-Oph. (B) and (E) are mean ± SEM from 3 independent experiments. *P < 0.05; ** P < 0.01; *** P < 0.001 determined by one-way ANOVA.

Article Snippet: NTC or APOL3 -/- HeLa cells were transfected with pCLBW cox8-EGFP-mCherry (Addgene #78520) a gift from David Chan .

Techniques: Inhibition, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: The antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction

doi: 10.1101/2025.05.16.654477

Figure Lengend Snippet: (A and B) Cardiolipin immunofluorescence (single z planes) in IFN-γ-primed wildtype HeLa cells (+/- LLOME) permeabilized with saponin or Trition-X100 (A) or in NTC and PLSCR3 -/- cells + LLOME using saponin permeabilization (B). Quantification represents mean ± SEM from 3 independent experiments (n = 20 cells per replicate). Immunoblots (IB) are representative of 2 experiments. (C) Representative Airyscan image of IFN-γ-primed LLOME-treated APOL3 mnGFP cells stained for OMP25 with JF646 and CL. Single z slices and line profile is shown. (D) APOL3-GFP expressed in IFN-γ-primed NTC or PLSCR3 -/- cells treated with LLOME and immunostained for Tom20 and Lamp1. Fractional overlap for APOL3 is shown (n = 12 cells, representative of 3 independent experiments, error bars ± SD). Maximum intensity projections of Thunder deconvolved z stacks are shown. (E) qPCR quantification of mtNDA in the cytosol versus WCE in IFN-γ-primed HeLa genotypes treated with LLOME. Shown are floating bars (mean, min/max, n = 5 independent experiments). (F) Mitochondria isolated from NTC or PLSCR3 -/- cells treated with rAPOL3 +/- rBAX and mtDNA release detected by PicoGreen fluorescence. Data are mean ± SEM (n = 3). (G, H, I) Primary human airway epithelial cells (AECs) primed with IFN-γ (or not) were treated with TiO2 nanoparticles (24 hr) and IFN-β measured in the supernatants by ELISA (G). AECs were transduced with lentivirus encoding Cas9 and sgRNAs targeting APOL3 , PLSCR3 , or cGAS or cultured with ddC prior to treatment (H). Alternatively, AECs were stained with Tetramethylrhodamine Methyl Ester (TMRM) after 12 hr to probe mitochondrial membrane potential (ΔΨm) and quantified by flow cytometry (MFI: median fluorescence intensity). (G, H, I) represent the mean ± SEM with AECs prepared from three different organ donors. * P < 0.05; ** P < 0.01; *** P < 0.001 determined by one-way ANOVA (B, E, G, H, I) or two-tailed unpaired t test (D).

Article Snippet: NTC or APOL3 -/- HeLa cells were transfected with pCLBW cox8-EGFP-mCherry (Addgene #78520) a gift from David Chan .

Techniques: Immunofluorescence, Western Blot, Staining, Isolation, Fluorescence, Enzyme-linked Immunosorbent Assay, Transduction, Cell Culture, Membrane, Flow Cytometry, Two Tailed Test